The rational synthesis of peptides has been a challenging task that has been solved only in the past few decades. The principal reaction in peptides synthesis is acylation of the amino group of one amino acid by the carboxyl group of another amino acid to form an amide bond. Since each amino acid has both an amino group and a carboxyl group, a direct approach leads to the formation of many different peptides. A general method that has been developed to avoid these difficulties involves the use of protecting (or blocking) groups, i.e., the use of certain groups to attach to the amino group or the carboxyl group of the amino acid and render the amino group or carboxyl group unreactive while still permitting the desired reaction to take place. Thus, the successful formation of the desired amide bond requires activation of the functional groups that participate in the bond forming reaction and protection of those that are not involved. Both amino and carboxyl protecting (or blocking) groups have been developed.
The carboxyl groups of amino acids are normally protected by conversion to an ester. Since esters are hydrolyzed more easily than amides, the protecting group can be removed readily by alkaline hydrolysis. Although simple methyl or ethyl esters have been used, benzyl and tert-butyl are most commonly employed because each of the latter can be removed under a variety of conditions. For example, the benzyl ester can be removed not only by alkaline hydrolysis but also by catalytic hydrogenation, Li/NH.sub.3 reduction, or HBr/AcOH. The teft-butyl ester is significantly more stable to base and nucleophiles, but it can be readily cleaved under acidic conditions, such as with trifluoroacetic acid (TFA) alone or in organic solvents.
Protection of the amino group is a much more challenging task. Early efforts used the p-toluenesulfonyl (tosyl or Ts) group as the protecting group (E. Fischer, Chem. Ber., 48 (1915) 93). Sodium in liquid ammonia, for example, was used to cleave the Ts group (du Vigneaud et al., J. Am. Chem. Soc. 76 (1954), 3313). These methods are associated with a number of problems and find little use in modern peptide synthesis. Later efforts used alkoxycarbonyl protecting groups, e.g., the benzyloxycarbonyl (Cbz) and the t-butoxycarbonyl (Boc) groups. The Cbz group is readily introduced, and can be removed under a number of mild conditions, such as hydrogenolysis, Na/NH.sub.3 reduction, etc. The Boc group is also readily introduced and can be cleaved with HCl in organic solvents, neat TFA or TFA in organic solvents. Still more recently, 9-fluorenylmethoxycarbonyl (Fmoc) group has found widespread applications in both solid-phase and solution peptide synthesis. The Fmoc group is remarkably stable to acidic conditions, but can be readily cleaved with secondary amines via a .alpha.-elimination mechanism. A problem with the use of the Fmoc group, however, is its high cost and large molecular weight which limits its use for large scale application.
To activate the N-acylamino acids for peptide coupling, they are converted to acid chlorides. A problem with acid chlorides derived from N-acylamino acids is that racemization is observed in the peptide coupling step because they undergo facile cyclization to the easily racemized oxazolinones. Although nitrogen protection using the alkoxycarbonyl protecting groups improves amino acid chloride stability in comparison to other acyl groups (e.g., formyl, acetyl, benzoyl), they can still cyclize to oxazolinones, especially if tertiary amines are present. Nonetheless, the Cbz, Boc and Fmoc groups remain the most popular amino protecting groups in peptide synthesis. Due to the success of alkoxycarbonyl protecting groups, sulfur- and phosphorus-based protecting groups have not been widely employed, although the use of arenesulfonamides as amine protective groups was recognized long ago (see, E. Fischer, supra). The known cleavage procedures suffer from harsh conditions and poor generality, and arenesulfonamide cleavage in the amino acid series has been difficult (see, e.g., Horner et al., Chem. Ber. 98 (1965) 3462; Kovas etal. J. Org. Chem. 31 (1996) 119; Rudinger et al., Helv. Chim. 56 (1973) 2216; Kestemont, Tetrahedron Lett. 32 (1991) 1425). Amino acid-derived benzenesulfonamides can be deprotected, but racemization occurs to the extent of 1.5-2.5% with typical amino acids (5% for phenylglycine). Kemp et al., Synthesis, 32 (1988), report the use of anthracene sulfonyl chloride as a protecting group for .beta.-amino acids. The reported reaction involves the actual synthesis of the desired .beta.-amino acid. Fukuyama et al., Tet. Lett. 36 (1995) 6373 report the use of p-nitrobenzenesulfonyl chloride as a protecting agent.
While the alkoxycarbonyl groups arguably possess very desired properties as an amine protecting group (i.e., ready introduction, crystallinity, stability under coupling conditions and multiple methods of removal), the racemization problem seems to be inherent in the alkoxylcarbonyl groups because the possibility of oxazolinone formation is always present. Oxazolinone derivatives are much more prone to racemization, especially in the presence of an amine base, which is often used as an additive. At the same time, sulfur-based protecting groups have met with limited success, and further, racemization can also occur. Despite recognition and study of various aspects of the problem, the prior art has produced very little in way of the introduction of a readily removable amine protective group in which removal can occur in relatively mild conditions and without racemization, i.e., an N-protected amino acid halide that does not participate in oxazolinone formation.